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AOAC Final Action Method 2016.02 (Biotin)
Page 7 of 16
For gradient program see Table 2016.02B.
Table 2016.02B. Gradient Program
Time (min)
Flow Rate (mL/min) Mobile Phase A (%)
Mobile Phase B (%)
Mobile Phase C (%)
0.0
0.6
90
10
0
18.0
0.6
90
10
0
18.5
0.8
0
0
100
24.0
0.8
0
0
100
24.5
0.6
90
10
0
27.0
0.6
90
10
0
H. Quality Control
(a) Check system suitability by injecting Standard 3 five times. RSD should be
≤
2%.
(b) Run the calibration standards at the beginning and end of the sequence (slope drift
≤
2%).
(c) The six-point calibration should give a correlation coefficient
≥
0.997.
(d) Test one in five samples in duplicate. The duplicates should be within the method repeatability.
(e) Inject one of the calibration standards after every five sample injections.
(f) Analyze a reference sample (e.g., National Institute of Standards and Technology Standard
Reference Material 1849a) in duplicate.
(g) Identification of biotin peak is based on absolute retention time. Spectrum scan can be used for
peak purity confirmation if required.
(h) Perform three high level recoveries with every batch of immuno affinity columns
I. Calculation and Reporting
The chromatography software will automatically calculate the concentration of the sample in µg/100g,
provided the concentration of the standards in (µg/100mL), sample weight (g) and dilution are entered
correctly.
Manual calculation can also be performed by using the following equation:
Biotin or Biocytin (µg/100g) =
(Sample Area x Dilution)
(Slope x Sample weight in grams)
Dilution
= 10 (100 x 1 ÷ 10) Sample made up to 100mL, 10mL used for IAC clean-up to a final volume of
1mL with water for HPLC analysis. The dilution will be 20 if 5mL is used for IAC clean-up.
Slope
= Valid slope calculation based on concentration on X-axis and peak area on Y-axis
Sample weight
: Calculate powder equivalent in grams using the following equation for reconstituted
powder samples.
Sample weight (powder equivalent) in grams = (W1 x W3) / W2
For ready to feed liquid samples, the sample weight used for extraction is used for the calculation.
Report results to three significant figures, using microgram-per-100-gram units or convert to other units as
required.
J. Repeatability
The difference between the results of duplicate portions of the same sample tested at the same sequence
should not exceed 6% of the mean result.
K. Reproducibility
2016.02 (BIOTIN) FINAL ACTION REVIEW
FOR ERP USE ONLY
DO NOT DISTRIBUTE